藏猪源捷申病毒的检测和遗传演化分析

旨在调查四川地区藏猪捷申病毒（porcine teschovirus,PTV）的流行及分子特征和遗传演化。用RT-PCR法对2017-2018年采自四川省甘孜藏族自治州康定市14个藏猪场96份粪便样品进行检测,并对全基因组进行序列分析和基因分型。PCR检测结果表明,PTV核酸阳性率为20.8%（20/96,95% CI=13.2%~30.3%）,14个规模藏猪场中PTV猪场阳性率为57.1%（8/14,95% CI=28.9%~82.3%）,并从2份阳性样本中获得了2条近似全长的PTV全基因组序列,分别命名为SWU-E5-2018和SWU-ZG2-2018,核苷酸序列相似性为83.1%。通过VP1序列分析发现2条序列的血清型分别为3型和6型。通过基因重组分析发现SWU-E5-2018存在重组现象,重组区域位于衣壳蛋白基因P1的552-3 082 nt处。为了进一步研究2条PTV序列的演化过程,以贝叶斯进化分析软件进行分歧时间估算,结果表明,SWU-E5-2018的分歧时间约为2010年,SWU-ZG2-2018的分歧时间约为2011年。本研究首次对藏猪源PTV进行了初步调查,结果表明藏猪中存在一定程度的PTV感染,为深入研究藏猪源PTV遗传变异及其生物学特性提供了参考依据。     

四川省甘孜州藏猪戊型肝炎病毒分子流行病学调查

为了解甘孜州藏猪戊型肝炎病毒(HEV)的感染情况，分析其相似性和遗传进化关系。本试验用反转录套式聚合酶链反应(RT-nPCR)方法对采集于2018—2019年甘孜藏族自治州藏猪主要养殖区域4个县10个藏猪养殖场的229份藏猪粪便样本进行核酸检测，并对全部阳性样本进行ORF2基因部分片段扩增测序，遗传进化分析。结果显示：猪粪便HEV核酸阳性率为16.59%(38/229,95%CI=12.00%～22.10%),猪场HEV阳性率为70%(7/10,95%CI=34.8%～93.3%);38个ORF2基因片段彼此之间核苷酸序列相似性为76.1%～100.0%,推导的氨基酸序列相似性为79.6%～100.0%,系统进化树分析显示所有阳性毒株均为基因4型，且与人源HEV毒株亲缘关系近。结果表明甘孜州藏猪中存在一定程度的HEV感染，与人源HEV毒株遗传关系近，相关部门采取防控措施以降低HEV感染的公共卫生风险。     

基于S基因序列分析新型猪流行性腹泻病毒的遗传演化

最近，笔者实验室在青藏高原地区发现两种新亚型藏猪源猪流行性腹泻病毒（PEDV），为进一步调查新型PEDV是否在四川腹泻猪群中存在或流行，对实验室2018-2019年保存的116份猪腹泻粪便或肠组织样本进行PEDV的检测及其纤突蛋白基因（spike）分子特征研究。结果表明：腹泻样本的PEDV检出率为42.2%（49/116，95% CI=33.1%~51.8%），并获得了13条完整的S基因序列，全长为4 149~4 170 bp，序列相似性为94.2%~99.9%，其中SWUN-H3-CH-SCYA-2019的S基因与藏猪源新G1亚群PEDV的序列相似性高达97.0%~98.6%。遗传演化研究结果表明13株PEDV S基因划分为G1和G2大群，其中SWUN-H3-CH-SCYA-2019位于藏猪源新G1亚群；SWUN-19-CH-SCZY-2018、SWUN-4-CH-SCXC-2018、SWUN-1-CH-SCNJ-2019和SWUN-3CH-CH-SCZG-2019位于G2亚群中一个独立的分支，且与藏猪源新G2亚群毒株有着较近的亲缘关系。为了进一步研究13株PEDV的演化过程，以贝叶斯进化分析软件包（BEAST）进行分歧时间估算，结果表明SWUN-H3-CH-SCYA-2019的分歧时间约为2012.3年，早于藏猪源新G1亚群其余毒株的最早分歧时间（2015.7年）；SWUN-4-CH-SCXC-2018、SWUN-19-CH-SCZY-2018和SWUN-3CH-CH-SCZG-2019的分歧时间约为2014.2年，早于G2亚群的藏猪源毒株2014.7年，所有藏猪源PEDV的分歧时间均晚于四川毒株。本研究在四川地区首次发现了藏猪源PEDV，并且从毒株的分歧时间推断青藏高原的藏猪源PEDV来源于四川，为新型PEDV分子遗传进化的监测提供了依据。     

藏猪源捷申病毒的检测和遗传演化分析

旨在调查四川地区藏猪捷申病毒(porcine teschovirus, PTV)的流行及分子特征和遗传演化。用RT-PCR法对2017—2018年采自四川省甘孜藏族自治州康定市14个藏猪场96份粪便样品进行检测,并对全基因组进行序列分析和基因分型。PCR检测结果表明,PTV核酸阳性率为20.8%(20/96,95%CI=13.2%～30.3%),14个规模藏猪场中PTV猪场阳性率为57.1%(8/14,95%CI=28.9%～82.3%),并从2份阳性样本中获得了2条近似全长的PTV全基因组序列,分别命名为SWU-E5-2018和SWU-ZG2-2018,核苷酸序列相似性为83.1%。通过VP1序列分析发现2条序列的血清型分别为3型和6型。通过基因重组分析发现SWU-E5-2018存在重组现象,重组区域位于衣壳蛋白基因P1的552—3 082 nt处。为了进一步研究2条PTV序列的演化过程,以贝叶斯进化分析软件进行分歧时间估算,结果表明,SWU-E5-2018的分歧时间约为2010年,SWU-ZG2-2018的分歧时间约为2011年。本研究首次对藏猪源PTV进行了初步调查,结果表明藏猪中存在一定程度的PTV感染,为深入研究藏猪源PTV遗传变异及其生物学特性提供了参考依据。     

猪戊型肝炎病毒荧光定量PCR检测方法的建立及其分子流行病学

对已发表的戊型肝炎病毒(HEV)不同基因型毒株全序列进行分析,针对ORF3的保守区设计引物和探针优化建立了HEV荧光RT-PCR检测体系。分析表明该反应体系具有良好的扩增效率、特异性和稳定性,且检测灵敏度比普通RT-PCR方法高10～100倍。利用所建立的荧光定量PCR方法对采集自浙江及其周边上海、江苏等地区规模化猪场的样品以及HEV抗体阳性人血清样品进行了HEV核酸检测。其中,77份人血清样本仅有2份为HEV核酸阳性(2.6%)。而猪粪便样品阳性率则高达20.5%(56/273),且所调查的猪场均存在HEV感染。进一步的分子流行特征分析表明,浙江及周边地区的猪HEV毒株多为基因Ⅳ型,与其他地区的Ⅳ型HEV毒株同源性较高(83.4%～89.5%),但它们之间也存在较多的变异位点。猪源HEV毒株PJ-1则与多数Ⅲ型HEV毒株同源性较高(88%),进化分析也表明其为基因Ⅲ型。人血清样品中检测到的2份HEV毒株Human-1与Human-2与本地区Ⅳ型猪源毒株在分子进化关系上具有很高的亲源性,它们分布于同一分支内,而不构成独立分支。以上分析结果表明浙江及其周边地区猪HEV感染较为广泛,且以基因Ⅳ型毒株为主,但也存在...     

利用宏基因组学鉴定藏猪腹泻粪便病毒种群

为进一步了解藏猪腹泻粪便中病毒种群，从青藏高原地区16个藏猪场采集腹泻粪便样本146份，将样本混合成一个pool样，提取RNA反转录成cDNA，建库后利用TruSeq Illumina测序，对测序的序列进行整理分析及相关病毒的分子特征进行研究。数据分析结果表明：藏猪腹泻粪便样本病毒种群包括11个病毒科的19种病毒，主要以线性和环状的小DNA病毒为主，如猪粪相关环状病毒7型、猪腺病毒和猪博卡病毒（PBoV）等；与腹泻相关的病原有猪流行性腹泻病毒（PEDV）、猪圆环病毒2型（PCV-2）和牛病毒性腹泻病毒1型（BVDV-1）3种；与腹泻相关的新发病原有porcine bufavirus、rabovirus和pasivirus 3种。利用SOAP软件组装出猪细小病毒6型（PPV-6）、PCV-2、PBoV-2完整或接近全长的基因组和戊型肝炎病毒（HEV）完整的ORF2基因序列，系统发育结果显示PPV-6和PCV-2与参考毒株有较近的遗传进化关系，PBoV-2与参考毒株有较远的遗传进化关系，单独聚为一簇，可能是一个全新的基因型。为进一步调查PCV-2在藏猪腹泻粪便中的检出率，对采集146份样本进行检测，检出率为10.96%（16/146，95% CI：6.4%~17.2%），且与四川地区PCV-2流行株有较近的亲缘关系。本研究发现藏猪腹泻粪便中病毒种类复杂多样，且可能存在与其他动物和人交叉感染情况，具有重要的公共卫生学意义，也为藏猪腹泻病防控提供一定的理论依据。     

基于S基因序列分析新型猪流行性腹泻病毒的遗传演化

最近,笔者实验室在青藏高原地区发现两种新亚型藏猪源猪流行性腹泻病毒(PEDV),为进一步调查新型PEDV是否在四川腹泻猪群中存在或流行,对实验室2018—2019年保存的116份猪腹泻粪便或肠组织样本进行PEDV的检测及其纤突蛋白基因(spike)分子特征研究。结果表明:腹泻样本的PEDV检出率为42.2%(49/116,95%CI=33.1%～51.8%),并获得了13条完整的S基因序列,全长为4 149～4 170bp,序列相似性为94.2%～99.9%,其中SWUN-H3-CH-SCYA-2019的S基因与藏猪源新G1亚群PEDV的序列相似性高达97.0%～98.6%。遗传演化研究结果表明13株PEDV S基因划分为G1和G2大群,其中SWUN-H3-CH-SCYA-2019位于藏猪源新G1亚群;SWUN-19-CH-SCZY-2018、SWUN-4-CH-SCXC-2018、SWUN-1-CH-SCNJ-2019和SWUN-3CH-CH-SCZG-2019位于G2亚群中一个独立的分支,且与藏猪源新G2亚群毒株有着较近的亲缘关系。为了进一步研究13株PEDV的演化过程,以贝叶斯进化分析软件包(BEAST)进行分歧时间估算,结果表明SWUN-H3-CH-SCYA-2019的分歧时间约为2012.3年,早于藏猪源新G1亚群其余毒株的最早分歧时间(2015.7年);SWUN-4-CH-SCXC-2018、SWUN-19-CH-SCZY-2018和SWUN-3CH-CH-SCZG-2019的分歧时间约为2014.2年,早于G2亚群的藏猪源毒株2014.7年,所有藏猪源PEDV的分歧时间均晚于四川毒株。本研究在四川地区首次发现了藏猪源PEDV,并且从毒株的分歧时间推断青藏高原的藏猪源PEDV来源于四川,为新型PEDV分子遗传进化的监测提供了依据。     

规模化猪场戊型肝炎病毒感染状况的调查与分析

为调查规模化猪场中戊型肝炎病毒(HEV)感染和流行特征,首先在4个猪场采集血样80份,用酶联免疫吸附试验(ELISA)榆测抗-HEV IgG抗体.在此基础上,作者于2007-2009年采集规模化猪场肛拭子样品415份,及规模化猪场送检的临床病猪肝脏样品135份,应用巢式PCR(RT-nPCR)方法进行HEV RNA检测,将PCR扩增产物测序,利用Neighbour-joining法进行进化分析.结果:4个猪场80份血清中HEV阳性血清56份,抗-HEV抗体阳件率高达60.0%以上.8个规模化猪场中有5个猪场的肛拭子检测到HEV,肛拭子阳性率8.43%(35/415);从临床送榆的发病猪采集的肝脏样品中HEV阳性率为15.55%(21/135);HEV RNA最早检出日龄为7 d,最晚检出日龄为84 d.遗传进化分析表明:测序的38株阳性毒株之间同源性为92%～100%,均属于HEV基因4型.其中,从肛拭子中分离的毒株(FJ445406)与猪源毒株DQ279091株处在同一分支上;而肝脏中分离的毒株(GQ202266.1)和人源毒株处在一个亚支,属于4型巾的同一亚型,表明该毒株与人源毒株亲缘关系较近.本研究表明华中地Ⅸ猪群中普遍存在HEV感染,阳性率较高.本研究为规模化猪场HEV的防控提供了依据.     

云南省部分猪场猪戊型肝炎的流行情况调查

为了解云南省猪群戊型肝炎感染情况,从云南省8个猪场采集166份新鲜猪粪便样品,用套式RT-PCR技术对戊型肝炎病毒(HEV)核酸进行检测,结果 21份样品阳性,阳性率为12.7%,感染率较高。通过PCR产物片段的测序分析,去除重复序列后得到11株基因序列。同源性分析结果表明,这11株毒株序列与选定的Gen Bank中基因1型、2型、3型和4型参考序列的核苷酸序列同源性分别为85.6%~87.8%,85.6%~87.8%,85.6%~95.6%和92.8%~98.9%,说明这些株毒株属于基因4型,与中国其他地区分离的毒株同源性最高,证实HEV的流行具有地理分布的特征。     

四川地区养猪场猪细小病毒病的病原检测及遗传进化分析

为了解四川地区猪细小病毒在猪群中的感染情况,对四川地区存在母猪流产情况的猪场进行抽样检测,采集10个猪场的20个样本,提取核酸,利用PCR方法进行猪细小病毒特异性片段的扩增,并选取阳性产物进行测序和序列分析.结果显示,检测样本中猪细小病毒阳性率为40％(8/20),序列分析发现,猪细小病毒阳性样本与吉林长春毒株DJH2...     

High prevalence of Hepatitis E virus in French domestic pigs

The importance of the domestic pig reservoir for Hepatitis E virus (HEV) was assessed by estimating the seroprevalence and prevalence of HEV contaminated livers in French slaughter-aged pigs. 6565 sera and 3715 livers were randomly sampled from 186 pig farms throughout the country. Taking the sampling design into account, the farm-level seroprevalence was 65% (95% CI 57–74) and 31% (95% CI 24–38) of the slaughter-aged pigs had antibodies against HEV. The individual prevalence of HEV RNA positive livers was 4% (95% CI 2–6) and 24% (95% CI 17–31) of the farms had at least 1 positive liver. Most isolates were of genotype 3f (76.7%) with smaller amounts of 3c (18.6%) and 3e (4.6%). The high prevalence of HEV in pigs and the similarities between HEV subtypes from pigs and humans corroborates the possible zoonotic origin of some HEV autochthonous infections.     

Detection and Characterization of Hepatitis E Virus in Domestic Pigs of Different Ages in Portugal

This study represents the primary hepatitis E virus (HEV) surveillance in domestic pigs in Portugal, five pig farms were investigated in 5 different Portuguese regions, ten faecal samples were collected at four different stages of the production. All faecal samples were tested for hepatitis E virus by real-time RT-PCR. At least one sample from each farms of all age groups tested positive for HEV. The prevalence in the pig herds varied from 10% to 30% and the mean prevalence was 32% in weaners, 20% in growers, 32% in fatteners and 4% in adult dry sows. Phylogenetic analysis of the detected HEV sequences indicated that the circulating virus strains belong under the genotype 3.     

Genotype 3 hepatitis E has been widespread in pig farms of Shanghai suburbs

Hepatitis E virus (HEV) genotype 3 was first identified in swine raised on a Shanghai suburban pig farm in late 2006. To accurately determine the prevalence of HEV infections among Shanghai pig farms, 426 pig fecal samples were collected from 37 pig farms located in all 10 Shanghai suburban districts and tested for the presence of HEV RNA using RT-PCR. Genetic analysis based on an amplified 150-bp ORF2 fragment revealed 111 samples to be HEV positive, and the prevalence of HEV infection within the different districts varied between 0 and 41.7%. Thirty-two samples were sequenced, and phylogenetic analysis indicated that 10 isolates belonged to HEV genotype 4 and were most closely related to 3 human and 2 swine HEV strains, all of which had originally been isolated from Asian countries including Japan and China. The remaining 22 isolates belonged to genotype 3 and were most closely related to a strain of swine HEV, US-SW, isolated from pigs in the United States. Our data indicated that genotype 3 HEV was widespread among suburban Shanghai pig farms although further study is required to determine the source and zoonotic nature of the virus.     

Retrospective serological study on hepatitis E infection in pigs from 1985 to 1997 in Spain

The objective of the present work was to ascertain the date in which hepatitis E virus (HEV) was introduced in the Spanish pig population. For this, a serological retrospective study was carried out using archived sera. A total of 2871 serum samples gathered between 1985 and 1997 and collected in 208 farms of Spain were tested for anti-HEV IgG by an in-house ELISA. Of the 2871 sera analyzed by ELISA, 1390 were positive for anti-HEV antibodies (48.4%, 95% CI: 46.9-49.9%) and that corresponded to 204/208 farms (98%, 95% CI: 96.1-99.9%) having at least one positive pig. Our results show that HEV was present and widespread in Spanish swine farms at least since 1985. Any significant changes in prevalence were detected from 1 year to another and therefore, HEV infection in swine should be considered endemic in Spain.     

Detection of hepatitis E virus shedding in feces of pigs at different stages of production using reverse transcription-polymerase chain reaction.

The aim of this study was to determine at which production stages hepatitis E virus (HEV) is shed by the highest number of pigs and to estimate the relative risk associated with each stage. For this purpose, 146 fecal samples of pigs from 21 farms were studied. In addition, 1 sample from the manure ditch and another sample of drinking water, collected directly from the trough located in the pen, were taken from 16 farms. HEV RNA was detected in fecal samples from 34 pigs (23.29%). The production stages in which most pigs excreted HEV were weaners (41.7%) and pigs in the first month of feeding (60%). The results of the statistical analysis showed that the principal significant risk stage in HEV shedding was the first month of feeding (odds ratio [OR] 19.5, 95% CI 3.59-106.07, P = 0.001) followed by the weaners stage (OR 9.3, 95% CI .78-48.42, P = 0.008). In 8 out of 16 farms tested (50%) HEV RNA was detected in raw manure and in the water trough of only 1. Detection of HEV in manure ditches raises the concern of how to deal with manure of swine origin, because it is used as soil fertilizer.